癌細胞之塑性已知會導致腫瘤的異質性，並進一步使癌症治療失敗。SOX2( SRY-box transcription factor 2)是一種參與幹細胞分化和重組的關鍵轉錄因子控制著癌細胞的塑性。細胞自噬作用是細胞分解代謝途徑的一種，在組織分化過程中調節代謝之可塑性。然而，SOX2之信息傳遞路徑是如何與細胞自噬作用相互影響以調節癌細胞塑性仍然模糊不清。在本篇研究中，我們發現了細胞自噬體結構蛋白 MAP1LC3A/LC3A 在肺腺癌中具有差異性的表達，其高表達與組織學分級和遠處轉移呈負相關。而一組的SOX2陽性和陰性的肺癌細胞分別顯示出不同的增生和侵襲的特性，我們將此細胞株用於研究SOX2信息傳遞路徑和細胞自噬作用之間的關聯性。 LC3A所介導的基礎細胞自噬作用在SOX2陽性的肺癌細胞中高度活躍，此細胞株具有高增生性和低侵襲性的特點。相比之下，SOX2陰性對應的細胞株則顯示出LC3A所介導的基礎細胞自噬作用較低，並伴有低增生性和高侵襲性的特徵。此外，與高侵襲性肺癌細胞相比，高增生性肺癌細胞表現更高的耗氧率(OCR)、更多的活性氧化物質(ROS)和更碎片化的粒線體。與對照組的肺癌細胞相比，抑制LC3A使肺癌細胞具有高侵襲性但低增生性的特徵，伴隨著較低的OCR、ROS 和較少碎片化的粒線體。SOX2能與LC3A的增強子結合，同時SOX2的過度表達增強了肺癌細胞中LC3A的表現。我們的研究結果提供了肺癌中差異表現的LC3A的新見解，用於調節細胞自噬作用和SOX2所介導的細胞增殖信息傳遞路徑，以控制粒線體的功能並影響肺癌細胞之塑性。

Cancer cell plasticity causes tumoral heterogeneity, leading to treatment failure in cancers. SOX2 (SRY-box transcription factor 2) , a key transcription factor involved in stem cell differentiation and reprogramming, controls cancer cell plasticity. Autophagy, a cellular catabolic pathway, regulates metabolic plasticity during tissue differentiation. However, how SOX2 signaling cross-talks with autophagy to regulate cancer cell plasticity remains elusive. Here, we report that MAP1LC3A/LC3A, an autophagosome structure protein, is differentially expressed in lung adenocarcinoma, and its high expression is negatively associated with histological grade and distant metastasis. Paired SOX2-positive and -negative lung cancer cells, which showed distinct proliferative and invasive properties, respectively, were used to study the crosstalk between SOX2 signaling and autophagy. LC3A-mediated basal autophagy was highly active in SOX2 positive lung cancer cells, which displayed high-proliferative and low-invasive characteristics. In contrast, SOX2 negative counterparts showed diminished LC3A-mediated basal autophagy accompanied with low-proliferative and high-invasive features. Furthermore, high-proliferative lung cancer cells exhibited a higher oxygen consumption rate (OCR), increased reactive oxygen species (ROS), and more fragmented mitochondria compared with their high-invasive counterparts. Silencing LC3A enriched lung cancer cells harboring high-invasive but low-proliferative features accompanied with lower OCR, ROS, and less fragmented mitochondrial pattern compared with the control lung cancer cells. SOX2 bound to LC3A enhancer, and SOX2 expression enhanced LC3A levels in lung cancer cells. Our findings provide novel insights of differential LC3A expression in lung cancer for regulating autophagy and SOX2-mediated proliferative signaling to control mitochondrial function and determine lung cancer cell plasticity.

1. Mizushima N. Autophagy: process and function. Genes Dev 2007;21:2861-73
2. Koukourakis MI, Kalamida D, Giatromanolaki A, Zois CE, Sivridis E, Poulilioul S, et al. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines. Plos One 2015;10
3. Bjørkøy G, Lamark T, Pankiv S, Øvervatn A, Brech A, Johansen T. Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1. Autophagy in Mammalian Systems, Part B2009. p 181-97.
4. Jia B, Xue Y, Yan X, Li J, Wu Y, Guo R, et al. Autophagy inhibitor chloroquine induces apoptosis of cholangiocarcinoma cells via endoplasmic reticulum stress. Oncol Lett 2018;16:3509-16
5. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006;10:51-64
6. Rabinowitz JD, White E. Autophagy and Metabolism. Science 2010;330:1344-8
7. Liu M, Jiang L, Fu X, Wang W, Ma J, Tian T, et al. Cytoplasmic liver kinase B1 promotes the growth of human lung adenocarcinoma by enhancing autophagy. Cancer Sci 2018;109:3055-67
8. Luo T, Fu J, Xu A, Su B, Ren Y, Li N, et al. PSMD10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with ATG7 and nuclear transactivation of ATG7 expression. Autophagy 2016;12:1355-71
9. Qu X. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. Journal of Clinical Investigation 2003;112:1809-20
10. Jing K, Lim K. Why is autophagy important in human diseases? Exp Mol Med 2012;44:69-72
11. Sabharwal SS, Schumacker PT. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? Nat Rev Cancer 2014;14:709-21
12. Mimaki M, Wang X, McKenzie M, Thorburn DR, Ryan MT. Understanding mitochondrial complex I assembly in health and disease. Biochim Biophys Acta 2012;1817:851-62
13. Vara-Perez M, Felipe-Abrio B, Agostinis P. Mitophagy in Cancer: A Tale of Adaptation. Cells 2019;8
14. Palikaras K, Lionaki E, Tavernarakis N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol 2018;20:1013-22
15. Wang Y, Liu HH, Cao YT, Zhang LL, Huang F, Yi C. The Role of Mitochondrial Dynamics and Mitophagy in Carcinogenesis, Metastasis and Therapy. Front Cell Dev Biol 2020;8:413
16. Westermann B. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Bio 2010;11:872-84
17. Youle RJ, van der Bliek AM. Mitochondrial Fission, Fusion, and Stress. Science 2012;337:1062-5
18. Serasinghe MN, Wieder SY, Renault TT, Elkholi R, Asciolla JJ, Yao JL, et al. Mitochondrial division is requisite to RAS-induced transformation and targeted by oncogenic MAPK pathway inhibitors. Mol Cell 2015;57:521-36
19. Kashatus JA, Nascimento A, Myers LJ, Sher A, Byrne FL, Hoehn KL, et al. Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth. Mol Cell 2015;57:537-51
20. Hamacher-Brady A, Brady NR. Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy. Cell Mol Life Sci 2016;73:775-95
21. He Y, Wu Z, Xu L, Xu K, Chen Z, Ran J, et al. The role of SIRT3-mediated mitochondrial homeostasis in osteoarthritis. Cell Mol Life Sci 2020;77:3729-43
22. Marusyk A, Polyak K. Tumor heterogeneity: causes and consequences. Biochim Biophys Acta 2010;1805:105-17
23. da Silva-Diz V, Lorenzo-Sanz L, Bernat-Peguera A, Lopez-Cerda M, Munoz P. Cancer cell plasticity: Impact on tumor progression and therapy response. Semin Cancer Biol 2018;53:48-58
24. Baylin SB, Jones PA. A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer 2011;11:726-34
25. Graf T, Enver T. Forcing cells to change lineages. Nature 2009;462:587-94
26. Zhang S, Cui W. Sox2, a key factor in the regulation of pluripotency and neural differentiation. World Journal of Stem Cells 2014;6:305-11
27. Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature Biotechnology 2008;26:1269-75
28. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76
29. Gontan C, de Munck A, Vermeij M, Grosveld F, Tibboel D, Rottier R. Sox2 is important for two crucial processes in lung development: Branching morphogenesis and epithelial cell differentiation. Developmental Biology 2008;317:296-309
30. Tompkins DH, Besnard V, Lange AW, Wert SE, Keiser AR, Smith AN, et al. Sox2 is required for maintenance and differentiation of bronchiolar Clara, ciliated, and goblet cells. Plos One 2009;4:e8248
31. Hussenet T, Dali S, Exinger J, Monga B, Jost B, Dembelé D, et al. SOX2 Is an Oncogene Activated by Recurrent 3q26.3 Amplifications in Human Lung Squamous Cell Carcinomas. Plos One 2010;5:e8960
32. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat Genet 2012;44:1111-6
33. Lu Y, Futtner C, Rock JR, Xu X, Whitworth W, Hogan BLM, et al. Evidence That SOX2 Overexpression Is Oncogenic in the Lung. Plos One 2010;5
34. Chou YT, Lee CC, Hsiao SH, Lin SE, Lin SC, Chung CH, et al. The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung cancer. Stem Cells 2013;31:2607-19
35. Chu YT, Yang PC, Yang SC, Shyu YC, Hendrix MJC, Wu R, et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American Journal of Respiratory Cell and Molecular Biology 1997;17:353-60
36. Hotta A, Cheung AY, Farra N, Vijayaragavan K, Seguin CA, Draper JS, et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat Methods 2009;6:370-6
37. Lin SC, Chou YT, Jiang SS, Chang JL, Chung CH, Kao YR, et al. Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell Plasticity. Cancer Res 2016;76:7036-48
38. Guo JY, Chen H-Y, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes & Development 2011;25:460-70
39. Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev 2011;25:717-29
40. Vallette FM, Olivier C, Lezot F, Oliver L, Cochonneau D, Lalier L, et al. Dormant, quiescent, tolerant and persister cells: Four synonyms for the same target in cancer. Biochem Pharmacol 2019;162:169-76
41. Hwang W, Chiu YF, Kuo MH, Lee KL, Lee AC, Yu CC, et al. Expression of Neuroendocrine Factor VGF in Lung Cancer Cells Confers Resistance to EGFR Kinase Inhibitors and Triggers Epithelial-to-Mesenchymal Transition. Cancer Res 2017;77:3013-26
42. Lee CJ, Sung PL, Kuo MH, Tsai MH, Wang CK, Pan ST, et al. Crosstalk between SOX2 and cytokine signaling in endometrial carcinoma. Sci Rep 2018;8:17550
43. Kauffman ME, Kauffman MK, Traore K, Zhu H, Trush MA, Jia Z, et al. MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS. React Oxyg Species (Apex) 2016;2:361-70
44. Hung GD, Li PC, Lee HS, Chang HM, Chien CT, Lee KL. Green tea extract supplementation ameliorates CCl4-induced hepatic oxidative stress, fibrosis, and acute-phase protein expression in rat. J Formos Med Assoc 2012;111:550-9
45. Chou YT, Hsieh CH, Chiou SH, Hsu CF, Kao YR, Lee CC, et al. CITED2 functions as a molecular switch of cytokine-induced proliferation and quiescence. Cell Death Differ 2012;19:2015-28
46. Xue YP, Kao MC, Lan CY. Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity. Sci Rep 2019;9:13694
47. Chen YC, Cheng TH, Lin WL, Chen CL, Yang WY, Blackstone C, et al. Srv2 Is a Pro-fission Factor that Modulates Yeast Mitochondrial Morphology and Respiration by Regulating Actin Assembly. iScience 2019;11:305-17
48. Glaab E, Garibaldi JM, Krasnogor N. ArrayMining: a modular web-application for microarray analysis combining ensemble and consensus methods with cross-study normalization. BMC Bioinformatics 2009;10:358
49. Sainz de Aja J, Dost AFM, Kim CF. Alveolar progenitor cells and the origin of lung cancer. J Intern Med 2020
50. Schlafli AM, Adams O, Galvan JA, Gugger M, Savic S, Bubendorf L, et al. Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer. Oncotarget 2016;7:39544-55
51. Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R, et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997;17:353-60
52. Huang SS, Huang JS. TGF-beta control of cell proliferation. J Cell Biochem 2005;96:447-62
53. Heldin CH, Landstrom M, Moustakas A. Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol 2009;21:166-76
54. Guo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM, et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev 2013;27:1447-61
55. Yun CW, Lee SH. The Roles of Autophagy in Cancer. Int J Mol Sci 2018;19
56. Sharifi MN, Mowers EE, Drake LE, Collier C, Chen H, Zamora M, et al. Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3. Cell Rep 2016;15:1660-72
57. Kenific CM, Stehbens SJ, Goldsmith J, Leidal AM, Faure N, Ye J, et al. NBR1 enables autophagy-dependent focal adhesion turnover. J Cell Biol 2016;212:577-90
58. Lock R, Kenific CM, Leidal AM, Salas E, Debnath J. Autophagy-dependent production of secreted factors facilitates oncogenic RAS-driven invasion. Cancer Discov 2014;4:466-79
59. Marsh T, Kenific CM, Suresh D, Gonzalez H, Shamir ER, Mei W, et al. Autophagic Degradation of NBR1 Restricts Metastatic Outgrowth during Mammary Tumor Progression. Dev Cell 2020;52:591-604 e6
60. White E. The role for autophagy in cancer. J Clin Invest 2015;125:42-6
61. Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 2006;5:14
62. Han SY, Jeong YJ, Choi Y, Hwang SK, Bae YS, Chang YC. Mitochondrial dysfunction induces the invasive phenotype, and cell migration and invasion, through the induction of AKT and AMPK pathways in lung cancer cells. Int J Mol Med 2018;42:1644-52
63. Trotta AP, Chipuk JE. Mitochondrial dynamics as regulators of cancer biology. Cell Mol Life Sci 2017;74:1999-2017
64. Rehman J, Zhang HJ, Toth PT, Zhang Y, Marsboom G, Hong Z, et al. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB J 2012;26:2175-86
65. van der Bliek AM, Shen QF, Kawajiri S. Mechanisms of Mitochondrial Fission and Fusion. Csh Perspect Biol 2013;5
66. Ferreira-da-Silva A, Valacca C, Rios E, Pópulo H, Soares P, Sobrinho-Simões M, et al. Mitochondrial Dynamics Protein Drp1 Is Overexpressed in Oncocytic Thyroid Tumors and Regulates Cancer Cell Migration. Plos One 2015;10:e0122308
67. Yu M, Nguyen ND, Huang Y, Lin D, Fujimoto TN, Molkentine JM, et al. Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer. JCI Insight 2019;5:126915
68. Yu M, Huang Y, Deorukhkar A, Fujimoto TN, Govindaraju S, Molkentine JM, et al. Mitochondrial Fusion Suppresses Pancreatic Cancer Growth via Reduced Oxidative Metabolism. bioRxiv 2018:279745
69. Lim SM, Mohamad Hanif EA, Chin SF. Is targeting autophagy mechanism in cancer a good approach? The possible double-edge sword effect. Cell Biosci 2021;11:56
70. Hao C, Liu G, Tian G. Autophagy inhibition of cancer stem cells promotes the efficacy of cisplatin against non-small cell lung carcinoma. Ther Adv Respir Dis 2019;13:1753466619866097
71. Liu F, Liu D, Yang Y, Zhao S. Effect of autophagy inhibition on chemotherapy-induced apoptosis in A549 lung cancer cells. Oncol Lett 2013;5:1261-5
72. Ren J-H, He W-S, Nong L, Zhu Q-Y, Hu K, Zhang R-G, et al. Acquired Cisplatin Resistance in Human Lung Adenocarcinoma Cells Is Associated with Enhanced Autophagy. Cancer Biotherapy and Radiopharmaceuticals 2010;25
73. Han W, Pan H, Chen Y, Sun J, Wang Y, Li J, et al. EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells. Plos One 2011;6:e18691